The practical utilization of regenerative medicine, which helps regenerate living tissues/organs that have fallen into functional disorder or functional incompetence, is currently proceeding. The regenerative medicine is novel medical technology of re-creating the same or similar forms or functions as in original tissues using 3 factors, i.e., cells, scaffolds, and growth factors, for living tissues that no longer recover by only natural healing ability intrinsically possessed by organisms. In recent years, treatments using cells have been being gradually realized. Examples thereof include cultured epidermis using autologous cells, cartilage treatment using autologous cartilage cells, bone regeneration treatment using mesenchymal stem cells, cardiac muscle cell sheet treatment using myoblasts, corneal regeneration treatment using corneal epithelial sheets, and nerve regeneration treatment. These novel treatments, unlike conventional alternative medicine based on artificial materials (bone prosthetic materials or hyaluronic acid injection), help repair or regeneration of living tissues and therefore produce high therapeutic effects. In fact, some products such as cultured epidermis or cultured cartilage using autologous cells have been launched.
In this context, for example, the regeneration of cardiac muscle using cell sheets is considered to require a multilayer construct of cell sheets for regenerating thick tissues. Okano et al. have recently developed cell sheets using a temperature-responsive culture dish. The cell sheets do not require treatment with an enzyme such as trypsin and thus retain cell-to-cell binding and adhesion proteins (Non Patent Documents 1 to 6). Such a cell sheet production technique is expected to be useful in the regeneration of cardiac muscle tissues (Non Patent Document 7). Okano et al. have also thought that a thickness of 200 μm or larger is impossible to achieve, and are developing cell sheets also containing vascular endothelial cells introduced therein in order to form vascular network in the cell sheets (Non Patent Document 8).
Also for bone regeneration, bone regeneration sheets comprising cultured cells added to matrices have been developed.
A bone regeneration sheet prepared by layering a cultured cell sheet comprising mesenchymal stem cells cultured into a sheet-like shape and a biodegradable sheet comprising biodegradable substances formed into a sheet-like shape (Patent Document 1) has been proposed. Moreover, there is a sheet for induction of mesenchymal tissue regeneration in which mesenchymal tissue precursor cells differentiated from mesenchymal cells and extracellular matrices are attached onto a porous sheet (Patent Document 2). Then, Patent Document 3 has reported that a sheet of 200 μm or larger in thickness can be formed by the development/optimization of a culture approach, and has also disclosed that the formation of approximately 210 μm cortical bone tissue layer was confirmed.
Furthermore, gel-embedding culture using collagen has been devised as one means of solving insufficient penetration of nutrients by only diffusion into a three-dimensional construct composed of cells (Non Patent Document 9). Moreover, Patent Document 4 states that three-dimensional culture is achieved by linking cells using inorganic ceramic beads.